Coronary heart disease is a major cause of mortality in the US and in our Veterans. Biological approaches to treat the diseased heart using cell delivery have shown only modest therapeutic benefit, in part due to poor cell survival and ineffective electromechanical coupling to the host myocardium. Our previous research demonstrates that delivery of therapeutic cells cultured in an extracellular matrix (ECM) can improve the survival and functionality of the transplanted cells, owing to the structural support of the ECM as a scaffold and the signaling cues they impart to the cells. In particular, we have previously demonstrated the potency of anisotropic nanofibrillar scaffolds in guiding cellular alignment along the direction of the nanofibrils, enhancing cell survival in ischemic tissues, and imparting signaling cues that confer cell function consistent with a non- diseased state. Anisotropic scaffolds may be well-suited for engineering cardiac tissue which also has highly organized cellular structure. In addition, induced pluripotent stem cells (iPSCs) may be a candidate cell source for the generation of autologous therapeutic cardiovascular cells. The long-term objectives of this research are to engineer a three-dimensional vascularized cardiac patch with pre-formed physiological cellular organization to repair ischemic heart disease; and to investigate the basic biological mechanisms underlying ECM-mediated cell-cell interactions that enhance cardioprotection under conditions of ischemia. We hypothesize that a three- dimensionally aligned iPSC-derived cardiac patch with endothelial interactions will provide more functional and viable engineered tissues for repair of myocardial infarction, due to more effective electrical coupling and organized tissue morphology, as well as the activation of cardioprotective nitric oxide signaling imparted by ECM nanopatterning. Accordingly, our specific aims are: 1) To engineer a vascularized aligned iPSC-derived cardiomyocyte (CM) patch and elucidating the molecular mechanisms of ECM-mediated nitric oxide signaling in enhancing iPSC-CM survival and phenotype. The iPSC-derived CMs (iPSC- CMs) and iPSC-derived endothelial cells (iPSC-ECs) will be co-cultured on three-dimensional oriented nanofibrillar collagen scaffolds with culture conditions optimized to promote iPSC-CM cell survival and function within the patch, when compared to patches that lack oriented nanopatterning or vascular interactions. The role of ECM-mediated nitric oxide signaling in enhancing iPSC-CM survival and phenotype will be investigated in the context of gain- and loss-of-function assays. 2) To determine the therapeutic effect of a vascularized aligned iPSC-CM patch for treatment of myocardial infarction. The vascularized oriented cardiac patch will be transplanted onto the epicardium of rats after myocardial infarction. The animals will be monitored over time for functional improvement in cardiac function, wall thickness, and electromechanical coupling. The role of nitric oxide in mediating the functional effects of the aligned vascularized cardiac patch will be quantified by measuring production of nitric oxide and related signaling molecules. The knowledge gained from these studies will provide a stronger foundation of basic knowledge and improved methods for clinical development of engineered cardiac patches, ultimately with the goal of restoring myocardial function to the diseased hearts of our Veterans.